Abstract
Background/Aim: Chemoresistance in rhabdomyosarcoma (RMS) is associated with poor survival, necessitating the development of novel anticancer drugs. Auranofin (AUR), an anti-rheumatic drug, is a thioredoxin reductase (TXNRD) inhibitor with anticancer properties. Although patient-derived xenograft (PDX) models are essential for studying cancer biology, reports on sarcomas using the PDX model are scarce because of their rarity. This study aimed to investigate the effectiveness of AUR treatment in RMS using a PDX model to evaluate its impact on local progression. Materials and Methods: A 20-year-old woman who was diagnosed with alveolar RMS was used to generate the PDX model. RMS PDX tumors were implanted in nude mice and divided into non-treated (vehicle) and treated (AUR) groups. Tumor volume and weight were evaluated, and immunohistochemical staining was performed to evaluate local progression of the sarcoma. The relationship between the TXNRD-1 expression and survival probability of patients with RMS was evaluated using publicly available expression cohorts. Results: AUR significantly suppressed RMS tumor progression over time. It also significantly suppressed the tumor size and weight at the time of excision. Histological evaluation showed that AUR induced oxidative stress in the PDX mouse models and inhibited the local progression of RMS by inducing apoptosis. High TXNRD-1 expression was found to be a negative prognostic factor for overall survival in patients with RMS. Conclusion: AUR-induced inhibition of TXNRDs can significantly impede the local progression of RMS through the oxidative stress-apoptosis pathway as demonstrated in PDX models. Thus, targeting TXNRD inhibition may be a promising therapeutic strategy for the treatment of RMS.
Soft tissue sarcoma is an extremely rare malignant tumor with a high incidence rate in adolescents and young adults (AYA) and is associated with poor survival (1). Rhabdomyosarcoma (RMS) is a major soft tissue sarcoma that is clinically and morphologically heterogeneous, accounting for 5-8% of all malignant tumors in children and AYA (2). The two most common histologic variants encountered in children and adolescents are the embryonal and alveolar subtypes (3). Effective treatment of RMS requires a multidisciplinary approach that encompasses surgery, chemotherapy, and radiotherapy. Although vincristine, actinomycin, and cyclophosphamide (VAC) treatment is an effective chemotherapy for RMS, chemoresistance is a frequent problem (4), necessitating the development of new anticancer drugs.
The cellular redox system involving oxidation and reduction reactions, regulates several vital cellular metabolic processes (5). Thioredoxin (TRX) and TRX reductases (TXNRD), the key molecules of the redox system, function as scavengers of reactive oxygen species (ROS) and are associated with the development of several diseases, including cancer (6). A recent report highlighted that the TRX inhibitor PX-12 and the TXNRD inhibitor auranofin (AUR) were highly effective in inhibiting the local progression and pulmonary metastasis of osteosarcoma (OS) in vivo (7, 8). This indicates that the redox system, which includes TRX and TXNRD, may be a valuable target for OS treatment. Habermann et al. reported that a combination of TRX and glutathione inhibitors, which are related to two main antioxidant pathways, is effective against RMS (9). However, further studies are required to fully understand the role of redox regulation in RMS. Patient-derived xenografts (PDX) are in vivo models that closely replicate essential tumor features and are used to study cancer biology and develop new therapies (10). Reports on sarcomas using the PDX model are scarce because of their rarity. Additionally, the redox system in the RMS PDX models has not been evaluated. Therefore, we constructed a PDX mouse model using tumor tissues from an RMS patient and investigated the effects of AUR, on local RMS progression in PDX mouse model.
Materials and Methods
Antibodies and reagents. Antibodies and reagents were obtained from the following commercial sources: AUR (SC-202476, Santa Cruz Biotechnology, Santa Cruz, CA, USA); Ki-67 (NCL-Ki67p, Leica BIOSYSTEMS, Milton Keynes, UK); 8-hydroxy-2′-deoxyguanosine (8-OHdG) (MOG-100P, JalCA, Shizuoka, Japan); Myoblast determination protein 1 (MyoD1), (CMQ 386R-16-RUO, Cell Marque, Rocklin, CA, USA); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (MK500, TAKARA BIO, Shiga, Japan); peroxidase-conjugated anti-rabbit IgG polyclonal antibody (#424144, Nichirei biosciences, Tokyo, Japan); peroxidase (POD)-labelled mouse immunoglobulin goat antibody (#414321, Nichirei biosciences).
Patients and PDX generation. The study was conducted in accordance with the principles of the Declaration of Helsinki, and all procedures were approved by the Institutional Review Board of Chiba Cancer Center (approval number: R04-021). Informed consent was obtained from the 20-year-old woman diagnosed with RMS before the start of the study. RMS tumor tissues were obtained via open biopsy. The soft tissue of the RMS obtained from the patients was transplanted into the quadriceps muscles of nude mice. The mice were examined every 3 days to confirm the implantation and growth of the tumor. Tumor size was evaluated 60 and 90 days after implantation. The resected tumor tissues were fixed with formalin and embedded in paraffin for hematoxylin and eosin (H&E) staining and MyoD1 immunostaining and the residual tumor tissues were stored in a cryogenic freezer (at −150°C) and were designated as 1st generation of PDX.
Animal studies. Six-week-old male BALB/cSLC nu/nu mice were obtained from Japan SLC (Shizuoka, Japan). The animals were housed in a controlled environment with a constant temperature of 22±2°C, humidity of 50±10%, and a 12-h light/dark cycle. All animal experiments were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of the Chiba Cancer Center. On day 0, a 2 mm wide cube of 1st generation RMS PDX was transplanted in the quadriceps muscle of 12 BALB/cSLC nu/nu mice. Primary tumor growth to a width of 5 mm was observed in 10 mice 60 days after PDX implantation.
In the current study, the success rate of implantation of PDX was 83%. The mice were divided into groups of five animals with similar distributions of tumor sizes. They were treated with either 200 μl vehicle control (40% PEG 300+60% sterile PBS) or 10 mg kg−1 AUR intraperitoneally, every day until the end of the study, as described in previous literature (8). Tumor measurements and animal weights were monitored weekly in a non-blinded manner. The volume was measured using a caliper and determined as length × width2/2. On day 49 after the initiation of the injection, the mice were anesthetized, and the tumors were removed for weight measurement and immunostaining. Tissues were fixed in formalin and embedded in paraffin. H&E staining and several immunostainings, including Ki-67, 8OHdG, MyoD1, and TUNEL, were performed to evaluate the local progression of sarcoma.
Immunohistochemical staining. H&E staining was performed as previously described (5). Other immunohistochemical staining was performed on formalin-fixed, paraffin-embedded 3 μm sections of tumor samples. Immunostaining was performed by incubating tumor sections with the primary antibodies Ki-67 (1/2,000), 8OHdG (1/1,000), and MyoD1 (1/250) at 4°C overnight. Next day, the tumor sections were incubated with secondary antibodies (POD-conjugated anti-rabbit IgG polyclonal antibody for Ki-67 and MyoD1 and POD-labelled mouse immunoglobulin goat antibody for 8OHdG) at 20°C for 30 min. TUNEL staining was performed using the in situ Apoptosis Detection Kit (TAKARA BIO), as per the manufacturer’s instructions. The percentage of 8-OHdG, Ki-67, and TUNEL-positive cells was determined by counting the number of stained cells in six randomly selected fields per slide at a magnification of ×200.
TXNRD-1 Gene Expression Analysis. We analyzed the relationship between TXNRD-1 expression and the outcomes of patients with RMS by examining publicly available microarray data (GSE167059). We downloaded the TXNRD-1 gene expression data from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl), which was accessed on October 7, 2023. We used the R2 web-based application to generate Kaplan–Meier survival curves. To determine the best point for survival analysis, we identified the expression value at which the statistical log-rank had the greatest separation of the survival curves.
Statistical analyses. Experimental data were expressed as mean±standard deviation with significant differences between mean values calculated using the Student’s t-test. p<0.05 was used as the criterion for statistical significance.
Results
Case presentation. A 20-year-old woman presented to our orthopedic outpatient department with a palpable mass in her right foot. The patient had no history of trauma or intense exercise, and her medical history was unremarkable. Physical examination revealed an 8×2 cm mass in her right foot. Magnetic resonance imaging revealed a massive tumor lesion on the right sole of the foot (Figure 1A). Furthermore, positron emission tomography-computed tomography showed multiple bone metastases, including the femur, ischium, scapula, tibia, and spine, as well as lymph node metastases (Figure 1A). An open biopsy showed alveolar RMS (Figure 1B), and preoperative chemotherapy, including a combination of vincristine, doxorubicin, cyclophosphamide (VDC) and VAC, and peripheral blood stem cell transplantation, was performed. Next, a wide excision of the RMS of the foot was performed, and pathological assessment revealed that the effect of chemotherapy was >90%. Postoperative intensity-modulated radiation therapy was administered to the spine, pelvis, and feet.
Case presentation. (A) Magnetic resonance imaging revealed a large tumor throughout the right foot. T2-weighted images of the foot showed high signal intensity compared to muscle (arrowhead). Positron emission tomography-computed tomography showed multiple bone metastases, including the femur, ischium, scapula, tibia, and spine, as well as lymph node metastases. (B) The specimen taken from the foot showed atypical cells with mildly enlarged and darkly stained round nuclei, proliferating in solid structures between connective tissues. The neoplastic cells tested positively for MyoD1. Scale bars=50 μm. MRI: Magnetic resonance imaging; PET-CT: positron emission tomography-computed tomography; H&E: hematoxylin and eosin; MyoD1: myoblast determination protein 1.
PDX construction. Sixty days after implantation, the tumor size was 5 mm, reaching 10 mm at 90 days after implantation (Figure 2A). It was designated as 1st generation PDX of RMS and confirmed to be equivalent to RMS tissue using H&E staining and MyoD1 immunostaining (Figure 2B).
PDX construction. (A) The soft tissue tumor of the quadriceps muscle in nude mice was evaluated at days 60 and 90 post-transplantation (arrowhead). (B) H&E and MyoD1 immunostaining of indicated PDX tumor equivalent to RMS tissue of the patient. Scale bars=50 μm. PDX: Patient-derived xenograft; H&E: hematoxylin and eosin; MyoD1: myoblast determination protein 1; RMS: Rhabdomyosarcoma.
Auranofin suppresses the local progression of rhabdomyosarcoma using PDX models. Next, we evaluated the efficacy of AUR on the local progression of RMS using a PDX model. There was no significant difference in body weight between the vehicle and AUR groups during the course of the study (Figure 3A). Based on the evaluation of tumor volume, AUR was found to significantly suppress tumor progression over time (Figure 3B). Furthermore, AUR significantly suppressed tumor size and weight at the time of excision (Figure 3C-E). Pathological evaluation revealed the presence of significantly higher number of 8-OHdG positive cells in the AUR group than in the vehicle group (Figure 4A and B). Furthermore, Ki-67 positive cells in the AUR group were significantly lower than those in the vehicle group (Figure 4A and B). The number of TUNEL-positive cells in the AUR group was significantly higher (Figure 4A and B).
AUR suppresses the local progression of RMS using PDX models. (A) There was no significant difference in body weight between mice receiving vehicle and AUR treatments during the course. (B) Based on the evaluation of tumor volume, AUR was found to significantly suppress RMS tumor progression over time. (C-D) AUR significantly decreased tumor size. (E) AUR significantly decreased tumor weight at the time of resection. *p<0.05 using the Student t test. AUR: Auranofin; RMS: rhabdomyosarcoma.
Histopathological analysis of the primary tumor. (A) H&E staining and immunohistochemistry of 8-OHdG, Ki-67, and TUNEL between the vehicle and AUR group. (B) Quantitative evaluation of positive cells for each staining. *p<0.05 using the Student t test. Scale bars=50 μm. H&E: Hematoxylin and eosin; 8-OHdG: 8-hydroxy-2′ -deoxyguanosine; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; AUR: auranofin.
High-level expression of Thioredoxin reductase-1 is a negative prognostic factor for overall survival in rhabdomyosarcoma patients. Using publicly available expression cohorts, we analyzed the expression levels of thioredoxin reductase-1 (TXNRD-1) and its correlation with overall survival probability in patients with RMS (Figure 5). High TXNRD-1 expression correlated with poor overall survival in the expression cohorts analyzed. This result strongly suggested that targeting TXNRD-1 may be a viable therapeutic option for treating RMS progression.
TXNRD-1 Gene Expression Analysis. High-level expression of TXNRD-1 is a negative prognostic factor for overall survival in RMS patients. Kaplan–Meier analysis using published array data from two independent sets of tumors analyzed using the R2 Genomics Analysis and Visualization Platform. p=0.044 using the log-rank test. TXNRD: Thioredoxin reductase; RMS: rhabdomyosarcoma.
Discussion
In the present study, we established an alveolar RMS PDX model. Furthermore, using this PDX model, we demonstrated that the TXNRD inhibitor AUR suppressed the local progression of RMS via apoptosis. The first-line treatments for RMS involve chemotherapy, surgery, and radiotherapy. Although VAC therapy and radiotherapy may be effective, some patients are resistant to chemotherapy. It is essential to evaluate the clinicopathological characteristics and prognostic factors of RMS as it sometimes has an unfavorable outcome. According to a report by Lo et al., patients with RMS who have pretreatment LDH levels >400 U/l and negative immunohistochemical staining for desmin or MyoD1 may have a poor prognosis (11).
Currently, many ongoing studies are exploring novel treatment strategies. Recently, significant progress was made in understanding the genetic and epigenetic factors associated with RMS (12). Numerous studies on RMS have identified new diagnostic and prognostic markers that have led to the development of novel therapeutic strategies (13). ROS have been identified as significant targets in high-throughput drug-screening assays (2). Oxidative stress is a condition characterized by increased ROS levels in cells. This can damage important cellular components, such as DNA, proteins, and lipids. Notably, ROS are not only associated with progression of cancer and sarcoma, but they also play a crucial genetic role in sarcoma development. In recent years, there have been reports of fusion genes and genetic mutations in children and adults with RMS (14, 15). Treating RMS also involves targeting the disease at a genetic level.
In this study, we demonstrated that RMS induces apoptosis through oxidative stress, as shown by TUNEL and 8-OHdG immunostaining. In line with this, Dachert et al. reported that ferroptosis plays a role in regulating tumor progression in RMS (16). Increased ROS production by a ferroptosis inducer has contributed to novel redox regulation-based treatment strategies for RMS. Further research on cell death mechanisms, such as apoptosis and ferroptosis in RMS, is required.
Although it is well-known that RMS is relatively sensitive to radiotherapy, it can also be resistant. Marampon et al. revealed the important role of redox balance in reducing the therapeutic efficacy of radiotherapy in RMS (17). They also identified nuclear factor erythroid 2-related factor 2 (NRF2), a key molecule in the Kelch-like ECH-associated protein 1 (Keap1)–NRF2 system, as a potential therapeutic target for RMS. Inhibition of NRF2 could be an effective treatment for RMS resistant to radiotherapy.
The redox system, which consists of TRX and TXNRD, has been linked to the progression and spread of several types of cancers (18). Redox imbalance triggers oxidative stress, leading to cancer cell death. Various anticancer drugs induce oxidative stress in cancer and sarcoma cells, resulting in cell death (19). The current study found that AUR induced oxidative stress in PDX mouse models, which inhibited the local progression in RMS by inducing apoptosis. Previous reports have shown that AUR alone significantly inhibited pulmonary metastasis in patients with OS (8). Moreover, a pilot study in dogs has shown that AUR improved overall survival when combined with standard chemotherapy (20). Considering the efficacy of AUR on OS and RMS, it is possible that AUR could be effective in the treatment of various bone and soft tissue sarcomas. It is also important to note that AUR is a safe oral drug for rheumatoid arthritis, developed more than 30 years ago and approved by the FDA.
It is crucial to conduct studies using PDX before conducting clinical studies in humans, since PDX models closely mimic the essential features of patients’ tumors. Although limited research has been carried out on PDX in RMS, a study by Manzella et al. found that a subgroup of RMS was highly sensitive to AKT inhibitors and that NOXA-BCL-XL/MCL-1, which are crucial molecules in the mitochondrial apoptotic cascade, could be targeted therapeutically to modulate the drug response in RMS using the PDX model (21, 22). However, the current study is the first to use PDX to evaluate redox regulation in RMS.
Study limitations. First, further evaluation of embryonal and alveolar RMS is necessary. Second, a thorough assessment of intracellular signaling is required. In the future, we plan to establish PDX-based cell lines and evaluate the redox signaling pathway of RMS.
Conclusion
Using a PDX model the present study demonstrated that AUR, an inhibitor of TXNRDs, effectively inhibited the local progression of RMS via the oxidative stress-apoptosis pathway. Therefore, inhibiting TXNRD could be a potential therapeutic approach for RMS treatment. Further research is required to clarify redox signaling pathways in PDX-based cell lines.
Acknowledgements
This work was supported by a JSPS KAKENHI grant (23K14608), The Mother and Child Health Foundation, Chiba Foundation for Health Promotion and Disease Prevention, Kato Memorial Bioscience Foundation, The Hamaguchi Foundation for the Advancement of Biochemistry, and Funds for Gold Ribbon Network. We would like to thank Editage (www.editage.com) for English language editing.
Footnotes
Conflicts of Interest
The Authors declare no conflicts of interest relevant to the content of this article.
Authors’ Contributions
H. K. and S. K. designed and performed the experiments, analyzed the data, and wrote the article. H. K., Y. H., S. O., and T. Y. provided conceptual advice.
- Received October 28, 2023.
- Revision received December 21, 2023.
- Accepted December 28, 2023.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
